Background. Resveratrol is a natural polyphenol that exhibits anti-inflammatory effects. The aim of this study was to investigate the effects of resveratrol treatment on epithelium-derived cytokines and epithelial apoptosis in a murine model of atopic dermatitis-like lesions.Material and Methods. Atopic dermatitis-like lesions were induced in BALB/c mice by repeated application of 2,4-dinitrofluorobenzene to shaved dorsal skin. Twenty-one BALB/c mice were divided into three groups: group I (control), group II (vehicle control), and group III (resveratrol). Systemic resveratrol (30 mg/kg/day) was administered repeatedly during the 6th week of the experiment. After the mice had been sacrificed, skin tissues were examined histologically for epithelial thickness. Epithelial apoptosis (caspase-3) and epithelium-derived cytokines  were evaluated immunohistochemically.Results. Epithelial thickness and the numbers of IL-25, IL-33, TSLP and caspase-3-positive cells were significantly higher in group II compared to group I mice. There was significant improvement in epithelial thickness in group III compared with group II mice (p < 0.05). The numbers of IL-25, IL-33, and TSLP-positive cells in the epithelium were lower in group III than in group II mice (p < 0.05). The number of caspase-3-positive cells, as an indicator of apoptosis, in the epithelium was significantly lower in group III than in group II mice (p < 0.05).Conclusion. Treatment with resveratrol was effective at ameliorating histological changes and inflammation by acting on epithelium-derived cytokines and epithelial apoptosis. Atopic dermatitis (AD) is a chronic inflammatory skin disease primarily affecting children. Its prevalence has been steadily increasing during the last decade in both developed and developing countries . AlthougThe pathogenesis of AD has not been clearly identified, but most information about its immunological features has been obtained in recent years. It was demonstrated that the epidermal cells in AD are unique in terms of both their barrier and immunological properties. The epidermis of patients with AD exhibits significant barrier disruption and prominent keratinocyte pathology. Keratinocytes are specialized epithelial cells in skin tissue that contribute to the initiation and maintenance of the inflammatory process in AD and are capable of producing, as well as responding to, various inflammatory mediators . InterleResveratrol is a naturally occurring polyphenol found in various types of fruits and vegetables, most notably in the skin of red grapes. Several studies indicated that it exerts various pharmacological effects, such as anticancer, antioxidant, antiangiogenic, and anti-inflammatory properties . In thisad libitum in a pathogen-free laboratory in the same department. All experimental procedures complied with the requirements of the Dokuz Eylul University Animal Care and Ethics Committee (Registration number:92/2013)Twenty-one 6–8-week-old male BALB/c mice weighing 18–20 g, purchased from the Department of Multidisciplinary Animal Laboratory, Dokuz Eylul University , were used in this study. The animals were kept in hygienic macrolane cages in air-conditioned rooms under 12-h light/dark cycles for the experiment. Food and water were provided The induction of AD by using DNFB was established based on previous research . DNFB wan = 7 per group), as follows: group I (control), group II (vehicle control), and group III (treatment with resveratrol) (The 21 BALB/c mice were randomly divided into three groups (eratrol) .The acetone and olive oil mixture was applied to shaved back of group I (control) without DNFB in the same manner. Atopic dermatitis –like lesions were induced in Group II (vehicle control) and group III (treatment with resveratrol).Resveratrol was given to group III (treatment with resveratrol) at a dose of 30 mg/kg/day for 7 days during the 6th week. Resveratrol was administered to each mice after dissolved in 100 µL dimethyl sulfoxide (DMSO) in group III . ResveraAnimals were sacrificed by an overdose of ketamine 24 h after the last drug administration, and dorsal skin samples were obtained for histomorphological analysis.Severity of dermatitis was estimated macroscopically at the end of 5th and 6th weeks. The following scoring procedure was applied:0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms. The dermatitis score was described as the sum of the scores for erythema/hemorrhage, edema, excoriation/erosion and scaling/dryness .Skin samples were placed in buffered formalin for light microscopic evaluation. After fixation, skin samples were embedded in paraffin for light microscopic evaluation and 5-µm serial sections were obtained with a rotary microtome . The samples were then stained with hematoxylin and eosin. Using these samples, general tissue features were examined and the thickness of the epithelium was measured. Photomicrographs were taken with an Olympus DP70 camera , which was adapted on an Olympus BX51 model microscope . The photomicrographs were taken randomly from five fields of each section. A counting frame was randomly placed four times on the image analyzer system monitor, epithelial thickness was measured , and the average was taken.2O2 for 5 min to inhibit endogenous peroxidase activity and then in normal serum blocking solution. Sections were incubated in a humid chamber for 18 h at 4 °C with IL-33 monoclonal antibody at 1:100 , IL-25 monoclonal antibody at 1:100 , TSLP monoclonal antibody at 1:100 , and anti-caspase-3 antibody at 1:100 . Sections were incubated with biotinylated IgG, followed by streptavidin conjugated to horseradish peroxidase for 15 min, each prepared in accordance with the manufacturer’s instructions . Sections were finally stained with diaminobenzidine , counter-stained with Mayer’s hematoxylin, and analyzed using a light microscope (All sections were incubated in a solution of 3% Hcroscope .i + 1), where i was equal to the intensity of immunohistochemical staining with a value of 1–4, and Pi was the percentage of epithelial cells stained with each intensity, varying between 0–100% , moderate (+ + ), strong (+ + + ) and very strong (+ + + + ) brown staining. Staining intensity was graded semiquantitatively using H-scores, which were calculated using the following equation: H-score =ΣPi . Normality of the distribution was assessed using the Kolmogorov–Smirnov test. The measurements followed a non-normal distribution, therefore non-parametric comparisons were made by the Kruskal–Wallis test. Pairwise comparisons were made using the Mann–Whitney All mice developed AD-like lesions with repeated DNFB challenge in both group II and III at the end of 5th week. The application of DNFB to the shaved back of mice firstly induced erythema and hemorrhage, then edema, erosion, excoriation, dryness and scaling appeared. Dermatits scores were not significantly different between groups.Treatment with resveratrol during 6th week resulted a decreased dermatitis score in group III that is significantly lower compared to group II . There wp < 0.05), indicating that the model for AD-like skin lesions had been successfully established (p < 0.05)  than in group I (20.28 ± 1.15 µm) (ablished  and 4. E < 0.05)  and 4.p < 0.05) (p < 0.05) (p < 0.05) (The number of IL-25 positive cells per field in the skin biopsy were significantly higher in group II than in group I ( < 0.05) . The num < 0.05) . The num < 0.05) . The num < 0.05) .The number of IL-25 positive cells were significantly lower in group III compared to group II in skin biopsy . IL-33 pT-cell-mediated inflammatory skin reaction, and accompanying keratinocyte apoptosis  (κB). It was thus suggested that NFκB may play a role in the transformation of environmental insults into inflammation in skin tissue  . Resveraβ1) . In a muβ1) . A recenn tissue . AlthougT-cells, Langerhans cells, basophils, eosinophils, and keratinocytes. Among these cell types, keratinocytes have attracted more attention in the pathogenesis of AD due to their roles in skin barrier function and their contribution to the initiation and maintenance of inflammation , which migrate into draining lymph nodes. These DCs initiate the differentiation of prime naive allergen-specific CD4 cells into Th2 lymphocytes, which secrete various cytokines . In thiser cells . The roler cells . The roler cells . In our er cells . In addier cells . Anotherell line . AgainstIL-33 is a member of the IL-1 cytokine family. Allergens, microbes, and pro-inflammatory cytokines can trigger the release of IL-33 from the epidermal barrier . Its recIL-25 is a member of the IL-17 cytokine family that is expressed in epithelial cells in response to proteases such as allergen proteases, trypsin, and papain. It was reported that administration of IL-17 to mice promoted allergic inflammation by inducing IL-4, IL-5, and IL-13 gene expression . Moreovein situ in lesional eczematous skin and patch-test lesions of AD (γ)-induced apoptosis in keratinocytes was increased in the skin of patients with AD compared with that in healthy subjects  cascade is considered the main pathway by which apoptosis is orchestrated. The most prevalent protease in the cell is caspase-3. This caspase is the central executioner caspase, which is responsible for the majority of the effects in cellular death . It was hreshold . In thisκB is a possible common pathway because this transcription factor both activates the cytokines involved in Th2 inflammation and regulates the genes affecting apoptosis (κB has been found to be increased in the epithelium in chronic inflammatory diseases such as asthma (κB signaling. Even we could not make a clear connection with T cell response and apoptosis, the inhibitor effects of resveratrol on NFκB expression might resulted anti-inflammatory and anti-apoptotic effects in this AD-like murine model. Moreover, γ and IL-12. However, we could not show the effect of resveratrol on the Th-1 derived cytokines which should be taken into account in future experimental studies. Second, two blind histologists evaluated epithelial thickness in order to avoid a potential bias in our study but a marker for cell proliferation such as Kİ-67 protein could give a more conclusive data in this aspect (There are some limitations to this study. First, although we demonstrated beneficial effects of resveratrol on inflammation and apoptosis, we could not reveal the molecular pathways by which resveratrol acts on the epithelium of AD-like lesions. Inhibition of the expression of NFpoptosis . In addis asthma . Ren et s aspect . Third, In conclusion, our data suggest that systemic resveratrol treatment exerts anti-inflammatory and antiapoptotic effects in a murine model of AD-like lesions. Although it is too early to draw definitive conclusions, our data indicate that resveratrol may be therapeutically beneficial to improve epithelium-derived allergic responses. Specifically, it may be effective at suppressing the very first step in inflammation.10.7717/peerj.1889/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1889/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.1889/supp-3Supplemental Information 3H scores.Click here for additional data file.10.7717/peerj.1889/supp-4Supplemental Information 4Epithelial thickness.Click here for additional data file.


The apparent diffusion coefficient (ADC) is a highly diagnostic factor in discriminating malignant and benign breast masses in diffusion-weighted magnetic resonance imaging (DW-MRI). The combination of ADC and other pictorial characteristics has improved lesion type identification accuracy. The objective of this study was to reassess the findings on an independent patient group by changing the magnetic field from 1.5-Tesla to 3.0-Tesla.This retrospective study consisted of a training group of 234 female patients, including 85 benign and 149 malignant lesions, imaged using 1.5-Tesla MRI, and a test group of 95 female patients, including 19 benign and 85 malignant lesions, imaged using 3.0-Tesla MRI. The lesion of interest was segmented from the raw image and four sets of measurements describing the morphology, kinetics, DW-MRI, and texture of the pictorial properties of each lesion were obtained. Each lesion was characterized by 28 features in total. Three classical machine-learning algorithms were used to build prediction models on the training group, which evaluated the prognostic performance of the multi-sided features in three scenarios. To reduce information redundancy, five highly diagnostic factors were selected to obtain a compact yet informative characterization of the lesion status.Three classification models were built on the training of 1.5-Tesla patients and were tested on the independent 3.0-Tesla test group. The following results were found. i) Characterization of breast masses in a multi-sided way dramatically increased prediction performance. The usage of all features gave a higher performance in both sensitivity and specificity than any individual feature groups or their combinations. ii) ADC was a highly effective factor in improving the sensitivity in discriminating malignant from benign masses. iii) Five features, namely ADC, Sum Average, Entropy, Elongation, and Sum Variance, were selected to achieve the highest performance in diagnosis of the 3.0-Tesla patient group.The combination of ADC and other multi-sided characteristics can increase the capability of discriminating malignant and benign breast lesions, even under different imaging protocols. The selected compact feature subsets achieved a high diagnostic performance and thus are promising in clinical applications for discriminating lesion type and for personalized treatment planning. There is a growing clinical interest in developing noninvasive tissue characterization methods that can be used early in the course of diagnosis to assess risk and to guide subsequent treatment by allowing clinicians to conduct a therapy on an individual ,2. MagneDCE-MRI, on the other hand, uses the serial acquisition of images during and after the injection of an intravenous contrast agent. It has been shown to reflect tumor vascularity and to achieve higher sensitivity than other imaging modalities in delineating invasive lobular carcinoma, which is not evident on conventional imaging ,13. DCE-A recognized weakness of both DCE-MRI and DW-MRI is their low specificity in discriminating between benign and malignant lesions (37–86%) -17; therDuring the image interpretation phase, well-trained and experienced radiologists are needed to read an MRI image. However, even well-trained experts may have high inter-observer variation rates, so computer-aided diagnosis (CAD) is necessary to help radiologists in detecting and classifying breast cancer . RecentlWe, together with other researchers, have shown that combining different modalities, such as DCE-MRI and DW-MRI, can dramatically increase the power in discriminating pathologically verified breast masses ,27-29. Fk-nearest neighbors (KNN) [However, these earlier studies mainly concentrated on patients collected under similar protocols. Therefore, the obtained prognostic models, as well as the selected prognostic factors, were not validated extensively. We conducted an independent validation study concerning breast mass discrimination on two patient datasets collected under different imaging conditions. We focus on evaluating the potential discriminatory power by integrating DCE-MRI with DW-MRI. Twenty-eight distinct features were estimated to comprehensively characterize the segmented mass. Three scenarios were analyzed to resolve three major concerns. 1) Does the high diagnostic power reported still hold in an independent validation study? 2) Does a full characterization of breast mass improve diagnostic performance? 3) Can a compact feature set achieve good diagnostic performance? Our studies have given positive answers to these three questions through extensive experiments using standard classification models including SVM -33, k-ners (KNN)  and Randrs (KNN) . FinallyThis retrospective study was approved by the institutional review board (IRB) and ethics committee of Sun Yat-sen University Cancer Center, China. Neither patient approval nor informed consent was required for review of medical records or images. Informed consent was signed and obtained from all patients before biopsy or surgery prior to procedures as a daily practice. This study consisted of two groups of patients with lesions detected on breast MR images. These data were collected at the Sun Yat-sen University Cancer Center. Between September 2008 and December 2011, a total of 234 consecutive female patients were enrolled in the first group (training group), including 85 benign and 149 malignant lesions. All of the patients in the training group underwent a breast MRI examination in a 1.5-Tesla system. The mean age of these women was 46 years (ranging from 18 to 78 years). Between January 2011 and December 2011, a total of 93 consecutive female patients with 18 benign and 75 malignant lesions were enrolled in the second group (test group). The patients in the test group underwent a breast MRI examination in a 3.0-Tesla system. The mean age of these 93 women was 45 years (ranging from 16 to 74 years).The breast MRIs were interpreted using assessment and breast density categories established by the American College of Radiology and reported in the Breast Imaging Reporting and Data System (BI-RADS) by two radiologists who had 3–10 years’ experience in breast imaging. The entire breast images, breast tissue or lesions were classified as per the following assessments: need additional imaging evaluation (category 0); negative (category 1); benign finding (category 2); probably benign finding with a recommendation for additional imaging or biopsy (category 3); suspicious (category 4); or highly suggestive of malignancy (category 5). All of these cases were selected by experienced radiologists based on the following inclusion criteria. 1) Multiple breast MRI imaging sequences, including T1- and T2-weighted images, pre- and post-contrast images, DCE-MRI and DW-MRI, can be loaded simultaneously. 2) Nodal or mass lesions on breast MRI classified as category 2–5. 3) All malignant (category 4–5) and probably benign lesions (category 3) on MR images were verified by open surgical biopsy or fine needle biopsy, and all benign lesions (category 2) on MR images were verified by biopsy or follow-up at least 2 years after MRI examination.Patients were excluded from the trial for any of the following criteria: 1) history of previous breast biopsy within a week or any therapy on breast lesions before MRI examination; 2) lesions not visible in any sequences on breast MRI imaging; 3) lesions classified as category 3–5 could not be verified by histopathology. Characteristics and histopathology of the lesions in the two groups are summarized in Table The patients in the training group underwent MRI in a 1.5-Tesla superconductive magnetic system . The patients in the test group underwent MRI in a 3.0-T superconductive magnetic system . A breast-specific 4-channel phased-array surface coil was used. The images consisted of axial cross-sectional and sagittal T2-weighted fast spin-echo, sagittal T1-weighted non-fat-suppressed, T1-weighted fat-suppressed DCE before and after contrast material administration, and DW sequences prior to gadolinium-based contrast material injection in axial orientation. DCE MR imaging data were acquired using an MRI-specific automatic power injector  to inject 0.1 mmol/kg body weight contrast medium gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA) with a hand venipuncture technique at a rate of 3 ml/s. Saline, 10 ml at 3 ml/s, was then injected to wash the tube.2, 5000/75 (repetition time msec/echo time msec), 5-mm section thickness, a 30 × 30-cm field of view, a 256 × 256 matrix, 0 mm section gap, and 130 sec acquisition time. DCE MRI was obtained using 3D Fast FSPGR pulse sequence, with repetition time msec/echo time msec of 5.5/2.6, a matrix of 288 × 288, and nine postcontrast acquisitions. Temporal resolution was 59 seconds per dynamic acquisition.For 1.5 Tesla MR imaging, DW-MRI was performed using single-shot echo planar imaging, fat suppression, b values of 0 and 800 s/mm2, 5400/86 (repetition time msec/echo time msec), 5-mm section thickness, a 30 × 30-cm field of view, a 192 × 192 matrix, 1 mm section gap, and 130 sec acquisition time. DCE MRI was obtained using a  FL3D sequence, with repetition time msec/echo time msec of 4.15/1.55, a matrix of 256 × 205, and nine postcontrast acquisitions. Temporal resolution was 270 seconds per dynamic acquisition.For 3.0 Tesla MR imaging, DW-MRI was acquired using a spin-echo echo-planar imaging, fat suppression, b values of 0 and 800 s/mmThe manual segmentation was first performed by an experienced radiologist and optimized by a two-step approach through which we incorporated fuzzy c-means clustering  and a grOnce a segmented lesion image was obtained, one can characterize its pictorial properties by using a standard technique for image analysis. In our study, four groups of features were designed to reflect the distinct characteristics of the mass images, including kinetics, morphology, texture and DW-MRI features.The morphological group of features is traditionally used in clinical practice and it mainly summaries the one-dimensional statistics. Eleven morphological features were estimated for each segmented lesion. The features of the group include compactness, spiculation, extent, elongation, solidity, circularity, entropy of radial length distribution, fractal, heterogeneity, area, and eccentricity. Texture features are widely used in the pattern recognition domain to assist in differentiating imaged objects automatically, such as natural scenes versus non-natural scenes. They have also been widely used to analyze breast cancer images to discriminate abnormalities from normal masses . FundameThe texture features included angular second moment, contrast, correlation, inverse difference moment, average of sum, variance of sum, entropy of sum, entropy, average of difference, variance of difference, entropy of difference, measurement of correlation 1 information, and measurement of correlation 2 information . ReadersBoth the early-phase enhancement (EPE) and the signal enhancement ratio (SER)  were estI0, Iinit and Ilast represent the signal intensity in pre-contrast, first post-contrast and last images, respectively.where The discrimination capability of ADC has been validated, and its expression is shown to be significantly lower in malignant tumors than in benign breast lesions or normal tissue in DW-MRI ,11,44,45features for each lesion. All the features obtained were extracted by two radiologists who had 10 years’ experience in interpreting breast MRIs. They were blind to the histological results. The status of breast masses enrolled in the study were all verified histopathologically, or confirmed in at least the following two years. The systematic pipeline, consisting of four steps including image segmentation, feature calculation, feature extraction and classification, is summarized in Figure The four groups generated 28 t-test to compare malignant with benign. A p-value of less than .05 was considered to indicate a significant difference. Software  was used for all data analysis.We first assessed the overall classification performance of each individual feature in classifying lesion types. For each individual feature, the best cut-off value with which to differentiate benign from malignant lesions was first estimated on the training group through analyzing the receiver operating characteristics (ROC). The best cutoff value was defined as the value corresponding to the highest average of sensitivity and specificity. This value was then evaluated on the test group to validate its diagnostic performance. To remove the bias due to different magnetic field levels as well as inter-observer interpretations, the two groups were normalized using a standard z-transformation. The area under the maximum likelihood-estimated binormal ROC curve (AUC) was used as an index of performance. Features whose AUC was larger than 0.5 were further analyzed using an independent-samples It has been shown by ours research as well as in earlier studies that an individual feature is less effective in the characterization of breast lesions than multiple features combined ,27-29,46Though each segmented lesion was fully characterized by multi-sided descriptions, a redundant feature set will inevitably result, and deteriorate classification performance. To alleviate this drawback, a recently reported method for feature selection, called the Local Hyperplane-based RELIEF (LHR) feature weighting scheme, can be used to select a subset of features with high prognostic values -49. The Among the 28 estimated features, eleven of them achieved large AUC (>0.5), as shown in Table We considered three scenarios when evaluating the classification performance of multi-sided features in combination on the dataset. In the first scenario (scenario 1), we tested whether entire features achieved superior performance to individuals or combinations during diagnostic classification. In the second scenario (scenario 2), we tested whether ADC still possesses a high prognostic value when the magnetic field changed from 1.5-Tesla to 3.0-Tesla. In the third scenario (scenario 3), we tested whether carefully selected features achieved superior or comparable diagnostic performance to the entire feature set. Three conclusions were drawn with respect to the three scenarios.The estimated feature groups described distinct characteristics of the breast lesions that thus had different discrimination powers. First, we investigated the discrimination power of each feature group individually and then compared them with their combinations. Since the morphological information was widely used in clinical practice, it was used as the borderline to compare with other feature groups. Different combinations of feature groups with morphological features were tested using the three classifiers and their average performance was also computed. The results are summarized in Table t-test shows that the two groups are statistically different , as shown in Table It has been reported that ADC is a very informative diagnostic variable -9. The AThe estimated features were redundant in characterizing the lesion masses and therefore reduced the prediction performance of the three classifiers. A feature selection method reported recently, called LHR, uses a highly diagnostic yet compact feature subset . The fivBoth 1.5 T and 3.0 T systems are widely used in clinical practice. Magnetic power can influence the imaging parameters, such as signal-to-noise ratio, contrast-to-noise ratio, spatial resolution, and sequence acquisition time. Whether these changes in imaging protocol can influence the diagnostic performance of the classification models is rarely reported. The current study aimed to fill the gap by building a prognostic model on the training group of 1.5-Tesla patients and test it on the test group of 3.0-Tesla patients. Our multi-parametric model provided a high accuracy both in the 1.5-Tesla and 3.0-Tesla group. The results after the three well-designed scenarios demonstrate that diagnostic performance can be dramatically improved by incorporating multi-sided characterizations of breast lesions in MRI. The ADC parameter in particular shows a close relationship with lesion malignancy due to a high cell density, caused by an increased fraction of signals from intracellular water. This parameter, when combined with morphology and enhancement kinetic features, can increase both the specificity and sensitivity in discriminating lesion types, and thus is a promising candidate to provide supplementary assessment of lesion status.Our study has some limitations. First, the databases of the 3.0-Tesla group were not large enough to allow the extraction of a strict statistical model. Considering that both 1.5-Tesla and 3.0-Tesla systems are widely used in clinical practice, it will be valuable to evaluate the diagnostic performance of MRI at 3.0-Tesla on larger sample sizes in future research. Second, the pictorial characteristics were estimated on 2D slices and currently we are working on 3D characterization of the lesions to obtain accurate volumetric measurements.The current study has highlighted the potential of combining DCE-MRI with DW-MRI to differentiate breast mass from normal via extensive validation. Our study demonstrates that diagnostic performance can be dramatically improved by characterization of breast lesions through the incorporation of multi-modalities of the MRIs, thus yielding better mass classification than with individually processed features of the two modalities. The ADC parameter is confirmed to have a high diagnostic value alone or in combination with other features and our analysis shows that its good performance is mainly due to improvements in specificity. Our study also reported a compact yet informative variable for diagnostic prediction that has the highest performance. This may be useful for building a CAD system combining of the ADC value, morphological, and DCE features to help radiologists in classifying breast lesions on MRI.The authors declare that they have no competing interests.HM conducted the statistical analysis of the data and drafted the manuscript. LZ and YX collected the patient data, helped to analyze the data with relevant medical literature and revised the manuscript. LL supported the study coordination and YP designed the experiment. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/366/prepubPictorial feature definitions. Detailed definitions of the texture and morphological features used to characterize the lesion.Click here for fileClassification models. A short introduction of the classification models used in our experiments.Click here for fileFeature selection model of LHR. A simple explanation of the feature selection model of LHR.Click here for file


Small noncoding regulatory RNA exist in wide spectrum of organisms ranging from prokaryote bacteria to humans. In human, a systematic search for noncoding RNA is mainly limited to the nuclear and cytosolic compartments. To investigate whether endogenous small regulatory RNA are present in cell organelles, human mitochondrial genome was also explored for prediction of precursor microRNA (pre-miRNA) and mature miRNA (miRNA) sequences. Six novel miRNA were predicted from the organelle genome by bioinformatics analysis. The structures are conserved in other five mammals including chimp, orangutan, mouse, rat, and rhesus genome. Experimentally, six human miRNA are well accumulated or deposited in human mitochondria. Three of them are expressed less prominently in Northern analysis. To ascertain their presence in human skeletal muscles, total RNA was extracted from enriched mitochondria by an immunomagnetic method. The expression of six novel pre-miRNA and miRNA was confirmed by Northern blot analysis; however, low level of remaining miRNA was found by sensitive Northern analysis. Their presence is further confirmed by real time RT-PCR. The six miRNA find their multiple targets throughout the human genome in three different types of software. The luciferase assay was used to confirm that MT-RNR2 gene was the potential target of hsa-miR-mit3 and hsa-miR-mit4. Mitochondria are known as the powerhouse of the cell; they are membrane bound organelles present in most eukaryotic cells. Most of the chemical reactions required in cellular respiration take place in the mitochondria. The numbers of mitochondria are generally varied from cell to cell and organism to organism. Mitochondria have their own genome  that is different from nuclear genome. Plant mitochondria genomes are relatively larger in size (180–2400 kb). In contrast, animal mitochondria harbor much smaller, highly compact, and gene dense genome. For instance, human mitochondrial genome carries 37 genes in a 16.5 kb small circular genome. Of these 13 specific protein products especially the subunit of the respiratory gene complex and the remaining 24 encode RNA products. However, there are more than 1000 proteins that have been identified in the mitochondria; they are mainly imported through the outer mitochondrial membrane by the translocase mitochondria complex enzyme or the oxidative folding pathway of the intermembrane space. Mitochondria genome imported 22 tRNA and is involved in the trafficking of different proteins through distinct molecular mechanisms [Mitochondrial DNA have two strands, one is guanine-rich which is known as heavy (H) strand and the other is a cytosine-rich light (L) strand. Only noncoding segment of mtDNA is the displacement loop (D-loop), a region of 1121 bp. The heavy strand has 12 of the 13 polypeptide-encoding genes which also contain 14 of the 22 tRNA-encoding genes and both rRNA-encoding genes , 6. ThesIn earlier studies, it has been shown that microRNA (miRNA) were identified in human mitochondria –21. ThesIn this study we have predicted pre-miRNA and mature miRNA and their identified targets in the mitochondrial genome. We have also constructed an interaction map that unravels the relationship between miRNA and mitochondrial genome. A complete flow chart of this study  is shown in  Homo sapiens mitochondrion was downloaded from NCBI (http://www.ncbi.nlm.nih.gov/) as a query NC-012920.1, which is 16,569 nt in length. The locations of mitochondrial genes (mtDNA) and functions are collected from MITOMAP, a human mitochondrial genome database (http://www.mitomap.org).The complete genome of Homo sapiens mitochondrion is screened using EMBOSS e-inverted program (http://emboss.bioinformatics.nl/cgi-bin/emboss/einverted) to identify the repeats of length ~120 nucleotides that are capable of forming hairpin stem-loop structures. The following parameters are used for analysis: gap penalty: 12, minimum score threshold: 10, match score: 3, mismatch score: −4, and maximum extent of repeats: 500 bp.This complete genome sequence ofTotal 110 sequences were used as an input for Mfold and RNAfold program for identification of hairpin like structures.http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) is maintained by Vienna RNA web server and used for prediction of minimum free energy structures and base pair probabilities from single RNA or DNA sequences. The RNAfold web server predicts secondary structures of single stranded RNA or DNA sequences. Default parameters were used for prediction of the miRNA.RNAfold program  was used to distinguish the real pre-miRNA from other hairpin sequences with similar stem-loops (pseudo pre-miRNA); a hybrid feature that consists of local contiguous structure-sequence composition, minimum of free energy (MFE) of the secondary structure, and P value for randomization test was used. Besides, a novel machine-learning algorithm, random forest (RF), was introduced. Analysis of the predicted sequence, MiPred, decides whether it is a real pre-miRNA-like hairpin sequence [sequence .MiRAlign. This software was used as a novel genome-wide computational approach to detect miRNA in animals based on both sequence and structure alignment; it has higher sensitivity and comparable specificity than other reported homologue searching methods. miRAlign follows two approaches; first, to be able to find distant homologs, miRAlign requires neither the sequence conservation of the whole pre-miRNA sequence nor the nearly perfect match of the 22 nt mature part but just assumes relatively loose conservation of the mature miRNA sequence. Secondly, more properties of miRNA structure conservation are considered. miRAlign introduces a structure alignment strategy and each single miRNA can be used as a query to do homology search [y search .Mireval. This software works in three steps. In the first step it finds miRNA precursor-like structure, in the second step it searches for sequence similarity with a known miRNA, and in the third step it searches for the sequence conservation. Input sequence will be folded and evaluated by an SVM classifier (triplet-SVM) and then blasted against the miRBase dataset on all organisms and significant similarities will be reported. It also reports input sequence on their corresponding genome and the conservation score of this genomic location will be downloaded from the Ensembl database [http://tagc.univ-mrs.fr/mireval/.database ; this soE-value cutoff of 10 and maximum number of hits of 100.All six miRNA are searched for similarity in miRBase, a microRNA repository . miRBaseδG in Kcal/mol), and composition were calculated by employing Oligo Calc: Oligonucleotide Properties Calculator [Physicochemical properties such as %GC content, %AU content, molecular weight , free energy  and BLAT can be found at nd=start , 30. BLAnd=start .2. The growth media were supplemented with specific growth factor: rhEGF, 0.5 mL; dexamethasone, 10 mL; FBS, 50 mL; and GA-1000, 0.5 mL . At 50–70% confluency myotube differentiation was induced by changing the media and growing cells in DMEM supplemented with only 2% of horse serum for 3–5 days until the myotubes were visible. The media were changed every day with fresh media; the myotubes were collected by trypsinization and centrifuged at 1000 ×g for 10 minutes.Human skeletal muscle myoblast cells (HSMN Lonza) were cultured in tissue culture flask at a seeding density of 3,500–4000 cells/cm at 37°C with 5% CO2). The supernatant was decanted and the cells were pelleted; this step was repeated for two times. Then cells were resuspended in homogenization buffer . Cells were homogenized and incubated for 10 minutes on ice. This homogenate is then centrifuged with 2 M sucrose solution at 1200 g for 5 min and supernatant is transferred to another tube. This step is repeated twice by transferring the supernatant to a new tube each time and discarding the pellet. Then centrifugation is carried out at 7000 ×g for 10 minutes. Mitochondrial pellet was resuspended in mitochondrial suspension buffer . Recentrifugation is carried out to repellet the mitochondria at 9500 ×g for 5 minutes.To isolate mitochondria human skeletal muscle myoblast cells were centrifuged at approximately 370 g for 10 minutes. Cells were resuspended in NKM buffer  and treated with RNase-free DNase I (Promega) for 1 h at 37°C. The total RNA was quantified with spectrophotometer, Nanodrop ND-1000, to assay the degree of enrichment of mitochondrial RNA and the risk of cytoplasmic contamination at the mitochondrial RNA and DNA level. miRNA were isolated from mitochondrial fraction by using miRNeasy kit , according to the manufacturer's protocol.µg of human cytosolic and enriched mitochondrial protein was loaded for each sample and the protein was transferred to PVDF membrane (Amersham Biosciences). The membrane was blocked in TBS with 0.1% Tween 20 (TBST) containing 5% nonfat dry milk for 1 hr. Then the membrane was incubated with primary antibody and kept at 4°C overnight. Membrane was washed with TBST for three times for 15 min and incubated with corresponding horseradish peroxidase (HRP) conjugated secondary antibody  for 1 hr. Again TBST wash was given for 15 min and the blots were visualized with chemiluminescent reagents.Human skeletal muscle cells and enriched mitochondrial skeletal cell pellets were lysed with ice-cold RIPA buffer containing protease inhibitor. After centrifuging at 12,000 rpm for 10 min at 4°C, protein was collected from supernatant and quantified by Bradford method (BIO-RAD) using multimode reader. 100 β-actin peroxidase (Sigma ref. A3854) and rabbit GAPDH. The Western blot protocol and the secondary antibodies used are described earlier   .To check quality and purity of mitochondrial fractionation, mitochondrial gene (CYTB) and nuclear gene (GAPDH) were used to measure both transcripts expression and DNA relative quantification . ReverseµL of RNA) were reverse-transcribed in 40 µL reaction and diluted 100-fold in the RT-PCR analysis using miRCURY LNATM Universal RT microRNA PCR kit (Exiqon). Negative control excluding enzymes from reverse transcription reactions was performed and profiled like the samples. Each PCR panel contains a PCR control and 3 PCR sets for reference gene. CP and melting curve analysis was done by using the Roche Light Cycler 480 software.The RT-PCR analysis was performed following the Exiqon protocol; three samples of mitochondrial extract  reverse transcriptase (Promega). Further amplification of cDNA was performed by PCR using gene specific primers.To estimate the human mitochondria mRNA level, total mitochondria rich human myoblast cell extract was used. 100  earlier . The amoSix novel miRNA identified were used for miRNA target identification with help of three types of software: miRanda, RNA22, and RNAhybrid, as described here.miRanda. miRanda (version 1.0b), available at http://www.microrna.org/microrna/home.do, a dynamic programming algorithm that relies on the sequence complementarity [S) ≥ 150.00, duplex free energy (ΔG) = −25.00 kcal/mol, and scaling parameter (w) = 3.00. The six human mitochondrial mature miRNA and complete mitochondrial genome were used as reference and query sequences, respectively, as input to miRanda. entarity , 33, wasentarity  and the RNA22. This software predicts microRNA targets which is maintained by IBM at https://cm.jefferson.edu/data-tools-downloads/rna22-v2-0/. RNA22 does not rely upon cross-species conservation, thus allowing the discovery of microRNA binding sites that may not be present in closely related species. It first finds putative microRNA binding sites in the sequence of interest and then identifies the targeting microRNA. RNA22 can identify putative microRNA binding sites without a need to know the identity of the targeting microRNA; this permits the identification of binding sites even if the targeting microRNA is not among those currently known [ly known .RNAhybrid. RNAhybrid is the online microRNA target prediction software available at http://bibiserv.techfak.uni-bielefeld.de/rnahybrid. It has some useful features; among these are the possibility to disallow G:U base pairs in the seed region and a seed-match speed-up, which accelerates the program by a factor of 8. The algorithmic core of RNAhybrid is a variation of the classic RNA secondary structure prediction. Instead of a single sequence that is folded back onto itself in the energetically most favorable fashion, RNAhybrid determines the most favorable hybridization site between two sequences [equences .Normal and mutated 3′ UTR of MT-RNR2 gene was subcloned using PCR based methods to generate the reporter vectors containing miRNA binding sites. Then this construct was inserted into the multiple cloning sites downstream of the luciferase gene in the psiCHECK-2 luciferase miRNA expression reporter vector.For the luciferase assay, HEK-293 cells were cultured to 70–80% confluence in 24-well plates and cotransfected with psiCHECK2-MT-RNR2-3′ UTR or psiCHECK 2-mut-MT-RNR2-3-UTR vector plus 100 mM anti hsa-miR-mit3 or hsa-miR-mit4 separately using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. The cells were incubated with transfection reagent/DNA complex for 5 h and refreshed with fresh medium containing 10% FBS. At 48 h after transfection firefly and renilla luciferase activities were evaluated using the dual luciferase reporter assay system (Promega) and the renilla luciferase activity was normalized to firefly luciferase activity.By an experimental in silico approach, total 268 inverted repeats are generated as an output of emboss program that are used for identification of ~120-nucleotide sequence. The number reduces to 110 sequences. The screened sequences were used for input for Mfold and RNAfold program for identification of hairpin like structures of the pre-miRNA. The number was further reduced and finally 6 mature and pre-miRNA were found suitable as pre-miRNA or miRNA candidates.http://dx.doi.org/10.1155/2014/206382.).   Six miRNA and their functions were detected in controlling myoblast proliferation and differentiation. Several miRNA were detected in robust and reliable fashion in the mitochondrial RNA extracts.Total six human precursors and mature miRNA were predicted with help of these types of software Figures . LocatioE-value. hsa-miR-331-5p has human tumor suppressor role in prostate cancer, leukemia, gastric cancer, liver cancer, Parkinson's disease, and so forth [ C. elegans. They have various roles including developmental one to inhibit cell proliferation in breast, colon, and hepatic cancers, lymphoma, and uterine leiomyoma [All six mature miRNA were used for similarity search. hsa-miR-mit-1 has shown one hit with human miRNA hsa-miR-331-5p with 59 score and 4.9 so forth , 37. Theeiomyoma . hsa-miReiomyoma . hsa-miRδG in Kcal/mol), and composition were calculated (Table S1) (Supplementary Data 1). %GC content of all six miRNA varies from 36% to 45% and %AU content from 55% to 64%. Molecular weight  and free energy  of six miRNA are also provided in the table. Moreover alignment results show that seed sequence of few miRNAs are perfectly aligned and some aligned sequences shows one or two base pair mismatches. These pre-miRNA and mature miRNA were mapped in the intergenic region of the mitochondrial genome. The existence of the pre-miRNA and mature miRNA was experimentally evaluated by the Northern blot analysis in the mtRNA of the skeletal myoblast.The size of the miRNA varied between 19 and 22 nucleotides. Physicochemical properties such as %GC content, %AU content, molecular weight , free energy  and cytoplasmic protein GAPDH . The amoIn addition, the relative ratios of mitochondrial transcripts from the CYTB gene and GAPDH transcripts were estimated by quantitative real time PCR. The mRNA level of CYTB was dramatically increased in the mitochondrial fraction in comparison to the cytoplasmic nuclear extracts. These results indicate a high abundance of mtRNA in the mitochondrial fraction which also suggest a very low level of contamination of genomic RNA in this fraction. The concentration of small noncoding RNA was measured relative to the human miRNA control lane. A lower level of expression in mitochondrial miRNA relative to human miRNA suggested that miRNA concentration is lower in mitochondria, but a significant amount of miRNA was also enriched in the mitochondrial fraction . RelativThe amount of six predicted miRNA and their precursors in human mitochondria was assayed by quantitative Northern analysis .μg) was used for each lane.The hybridization was conducted by probing with labeled predicted sequences. The mtRNA extracted from purified mitochondria appeared to be reliable and good source for miRNA profiling. The separate Northern blots with equal amount of mitochondrial RNA were subjected to electrophoresis and hybridized separately with six miRNA probes. The Northern blots were reprobed with 5S RNA for gel loading of each Northern blot. Three miRNA were found in considerable trace level, whereas the other three miRNA are in detected level, suggesting that expression level of three miRNA reaches to the minimum detectable level when equal level of RNA  and MT-RNR2 (16S). The binding region between 1671 and 3229 is the region for MT-RNR2 gene (16S), where three miRNA, hsa-miR-mit3, hsa-miR-mit6, and hsa-miR-mit4, are binding and making a cluster at 2403-2446 binding sites.Hence, these three miRNA may have role in regulating the activity of MT-RNR2 gene. MT-RNR2 gene (mitochondria encoded 16S RNA) encodes a mitochondrial ribosomal RNA (reran) in humans. The MT-RNR2 gene also encodes the Humanin polypeptide that has been the target of Alzheimer's disease research , 41, so Second miRNA cluster  is binding at 9184-9222 site which is the site for two genes, namely, MT-ATP6 (ATPase6∖ATP synthase F0 subunit 6) which lies in region 8527-9207 and second gene MT-CO3 (COIII∖cytochrome C oxidase subunit III) which lies in region between 9207 and 9990. MT-ATP6 is an ATP synthase.  It forms one part (subunit) of a large enzyme called ATP synthase. This enzyme, which is also known as complex V, is responsible for the final step of oxidative phosphorylation. It is found that mutations in the MT-ATP6 gene result in neuropathy, ataxia, and retinitis pigmentosa, which lead to muscle weakness, vision loss, and the other features. Cytochrome C oxidase is a unit of three complexes; cytochrome C oxidase subunit 3 is an enzyme that in humans is encoded by the MT-CO3 gene. Defects in MT-CO3 are a cause of Leber hereditary optic neuropathy (LHON). LHON is a maternally inherited disease resulting in acute or subacute loss of central vision, due to optic nerve dysfunction. Cardiac conduction defects and neurological defects have also been described in some patients. These three miRNA  may have role in regulating activity in neuropathy, ataxia, retinitis pigmentosa, and LHON.Comparison of miRanda and RNA22 has found that 20 miRNA targets share binding site similarity with each other. These 20 miRNA are mapped on mitochondrial genome with respect to their locations ; they coThe interaction map is plotted to show interrelation between genomic miRNA and mitochondrial genome. Total six miRNA are interacting with mitochondrial genome. This is shown with respect to output of three types of software, that is, number of miRNA predicted by the software. A typical arrow corresponds to type of software predicting the target and the number on that arrow represents number of miRNA targets predicted by the software .Total 28 diseases/disorders regions miRNA targets are identified by using miRanda and RNA22 software (Tables S4 and S5) (Supplementary Data 1), which shows miRNA binding sites in mitochondrial disease associated genes. In Table S4 28 diseases are listed with their genes. In Leber hereditary optic neuropathy (LHON) 10 genes are involved; these are MT-ND1, MT-ND4, MT-ND4L, MT-ND6, MT-CO1, MT-CO3, MT-CYB, MT-ND2, and MT-ND5. miRanda data has been explored for LHON; all genes are found to be under regulation of miRNA. In Leigh syndrome MT-ATP6, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, MT-ND6, MT-TK, MT-TV, and MT-TW are listed, in which 8 genes are under regulation of miRNA but genes MT-TK and MT-TV do not have any miRNA targets. MELAS syndrome is also under regulation of miRNA. Gene MT-RNR2 is located on mitochondrial genome at 1671-3229 bp position and codes for 16S ribosomal RNA. The MT-RNR2 gene also encodes the Humanin polypeptide that has been the target of Alzheimer's disease research and is found to be regulated on eight sites by two miRNA, namely, hsa-miR-mit4 and hsa-miR-mit3. Diabetes mellitus and deafness are associated with abnormalities in gene MT-TL1, which codes for tRNA leucine 1. This gene is under control of hsa-miR-mit3, which is binding at 3237-3260 target sites. Six diseases are not found to be under regulation of any miRNA; see Table S4 for more details. Table S6 (Supplementary Data 1) shows information of mitochondrial genes, position, and description. miRanda and RNA22 target intersection data is also explored for miRNA target intersection in all mitochondrial diseases. In LHON out of 10 genes 7 are found to be regulated by miRNA. MT-ND4L, MT-CO3, and MT-CYB are not controlled by any miRNA. In MELAS syndrome MT-TK, MT-TV, and MT-TW are not targeted by any miRNA. In Alzheimer's disease MT-RNR2 gene is under control of hsa-miR-mit3, hsa-miR-mit2, hsa-miR-mit3, and hsa-miR-mit4. MT-ND6 a diabetes and myopathy gene which is also under influence of hsa-miR-mit4 and hsa-miR-mit2 at 14344 to 14377 bp position; see Table S5 for more details (Supplementary Data 1).Intersection of data of all three types of software  shows that three genes are sharing common binding sites for these miRNA. MT-RNR2 gene, which is responsible for Alzheimer's disease, is also found to be under regulation of three miRNA, that is, hsa-miR-mit3, hsa-miR-mit6, and hsa-miR-mit4. MT-ATP6 is one of the important genes which encodes ATP synthase F0 subunit 6; fluctuation of this genes activity is associated with diseases such as LHON, Leigh syndrome, neuropathy, ataxia, and retinitis pigmentosa, NARP, and bilateral necrosis of striation. hsa-miR-mit3, hsa-miR-mit3, and hsa-miR-mit4 bind at region 9184-9222 on MT-ATP6 and control all the diseases associated with them. MT-CO3, which codes for cytochrome C oxidase subunit III, is responsible for diseases such as LHON and MELAS syndrome also under regulation of these miRNA. We believe that these miRNA which are binding at the same region control activity of genes in strong manner by way of competitive selection.A luciferase assay was performed to detect if MT-RNR2 gene (mitochondria encoded 16S RNA) was the direct target for hsa-miR-mit3 and/or hsa-miR-mit4 as described earlier . The expHuman miRNA were monitored by RT-PCR analysis of specific miRNA assays and controls. The analyses were further performed on three increasing quantities of the mitochondrial RNA input. The mtRNA extracted from purified mitochondria appeared to be a good source for miRNA profiling. The top-six most common detected miRNA in the mitochondria of the human myotubes were in homology with the following human genomic miRNA. We detected 6 miRNA known for their functions in controlling myoblastic proliferation and differentiation. The miRNA let-7 family was well represented. Many miRNA were detected in the mitochondrial RNA extract, so their potential targets in the mitochondrial genome should be investigated based on their location in the 3' end or interdomain of the gene.Cp < 35) was 43, 123, and 251, respectively , fourth has shorthand of genes and last fifth column has description of genes.


Microbial tolerance to different environmental stresses is of importance for efficient production of biofuels and biochemical. Such traits are often improved by evolutionary engineering approaches including mutagen-induced mutagenesis and successive passage. In contrast to these approaches which generate mutations in rapidly growing cells, recent research showed that mutations could be generated in non-dividing cells under stressful but non-lethal conditions, leading to the birth of the theory of stress-induced mutagenesis (SIM). A molecular mechanism of SIM has been elucidated to be mutagenic repair of DNA breaks. This inspired us to develop a synthetic SIM module to simulate the mutagenic cellular response so as to accelerate microbial adaptive evolution for an improved stress tolerance.Escherichia coli. The synthetic module enables expression and repression of the genes related to up- and down-regulation responses during SIM in a bistable way. Upon addition of different inducers, the module can be turned on or off, triggering transition to a mutagenic or a high-fidelity state and thus allowing periodic adaptive evolution. Six genes  in the up-regulation responses were evaluated for their potentials to enhance the SIM rate. Expression of recA, dinB, or ropS alone increased the SIM rate by 4.5- to 13.7-fold, whereas their combined expression improved the rate by 31.9-fold. Besides, deletion of mutL increased the SIM rate by 82-fold. Assembly of these genes into the SIM module in the mutL-deletion E. coli strain elevated the SIM rate by nearly 3000-fold. Accelerated adaptive evolution of E. coli equipped with this synthetic SIM module was demonstrated under n-butanol stress, with the minimal inhibitory concentration of n-butanol increasing by 56 % within 2.5 months.A controllable SIM evolution module was devised based on a genetic toggle switch in n-butanol tolerance of E. coli.A synthetic SIM module was constructed to simulate cellular mutagenic responses during SIM. Based on this, a novel evolutionary engineering approach—SIM-based adaptive evolution—was developed to improve the The online version of this article (doi:10.1186/s13068-015-0276-1) contains supplementary material, which is available to authorized users. The past decade has witnessed the power of synthetic biology in the microbial production of fuels and chemicals from renewable resources. Much effort has been directed towards the design and development of novel synthetic genes, pathways, circuits, and modules to improve productivity –4. HowevWe aimed to improve microbial stress tolerance by endowing cells with the capability of autonomous adaptive evolution when facing environmental stresses. This can be achieved by equipping cells with a synthetic evolution module. The evolution module was designed to increase the cellular mutation rate in response to stress signals, based on a new evolutionary theory of stress-induced mutagenesis (SIM). SIM experiments indicate that non-lethal stress will inhibit cell growth and trigger intracellular state transition to a transient hypermutation state in a small subpopulation (<0.1 %) of cells, thereby promoting adaptive evolution under stressful condition –10. ThisE response), and heat-shock response, and down-regulation of mismatch repair (MMR) , which is the model strain for quantitative SIM rate assay, constitutively expresses a lacI-lacZ fusion containing a +1 frameshift mutation on the F’ conjugative plasmid that renders it Lac− [+ revertants on lactose plates after incubation of 2 days is thought to be derived from spontaneous mutation during cultivation, whereas the gradual appearance of Lac+ revertants during prolonged incubation (3–6 days) is believed to be the consequence of SIM under lactose starvation [+ reversion within 3–6 days, which can be calculated by the total Lac+ colonies on day 6 minus those appeared on day 2 and then divided by 4 days [dinB), pTL03 (recA), pTL04 (rpoS), and pTL06 (nusA) increased the SIM rate by 4.5-, 13.7-, 5.7-, and 1.3-fold, respectively, compared with the control plasmid pTL01 . Therefore, only dinB, recA, and rpoS were selected to be SIM accelerators that needed to be up-regulated. Further gene-order optimization of these genes in pTL16 produced a highest SIM rate that was 31.9-fold greater than that of the control strain, E. coli FC40/pTL01.All pTL plasmids were transformed into  it Lac− . The raparvation . The raty 4 days . For thedinB, recA, and rpoS, while pML controlled the SIM decelerator gene, mutL. This SIM module was transformed into SMB07 , yieldi+ colonies increased almost linearly within 3–6 days of incubation with IPTG, showing a typical SIM profile. The FC40 strain showed a SIM rate of 0.4–0.5 Lac+ colonies per 106 cells per day in the presence of IPTG or aTc, which was comparable with the reported SIM rate of an independently constructed FC40 strain with araBAD deletion (0.5 Lac+ colonies per 106 cells per day) [mutL gene in SMB07 increased the SIM rate by 82-fold. Using the same control strain FC40, up-regulation of the genes dinB, recA, and rpoS in FC40/pTL16 elevated the SIM rate by 618-fold, although the empty pTL01 plasmid alone raised the SIM rate by 19-fold. However, the combination of mutL disruption and dinB-recA-rpoS expression in SMB07/pML/pTL16 only resulted in a 2923-fold increased SIM rate over FC40, which was much lower than the product of the individual increases contributed by the two parts (82-fold times 618-fold). One possible reason for this is the leaky expression of the PLtetO-1 promoter. As shown in Additional file LtetO-1-controlled gfp in medium without any inducer. Another possible reason might be the addition of 25 μg/mL of tetracycline to maintain the plasmid pML. Because of its similarity to aTc, tetracycline at this concentration might also serve as a weak inducer. Therefore, the expression of mutL and the repression of dinB-recA-rpoS might be induced to a certain degree, both of which restricted the increase of SIM rate.For all strains, the number of Lacper day) . Compare+ reversion mutation assay, the SIM module cannot be turned off to achieve the high-fidelity state, since lactose can induce the “SIM accelerator” genes under the control of the Ptrc-2 promoter in pTL16. To test whether aTc can turn off the module and switch the cell back to the high-fidelity state, the module was dissected into the “SIM decelerator” part (SMB07/pML) and the “SIM accelerator” part (pTL16), and the function of aTc on each part was separately tested.The results above clearly showed that IPTG turned on the SIM module and switched the cell to the mutagenic state. However, during the Lacgfp gene to construct plasmid pTLCG. The fluorescence of the aTc-induced strain SMB07/pML/pTLCG was less than 2 % of the IPTG-induced same strain  agar plates containing 10–12 g/L n-butanol  of SMB07/pML/pTL16 from 8 to 10 g/L, while another eight rounds further increased the MIC value to 12.5 g/L. In contrast, the control strain without the SIM module, FC40/pACYC184/pTAD, which underwent the same scheme, only reached an MIC of 10 g/L after ten rounds of evolution. In our previous work, the mutL-defective strain SMB07 improved the n-butanol MIC value of the parent strain from 9.5 to 13 g/L within 12 rounds of evolution [n-butanol MIC obtained by the SIM module was a little lower than that obtained by SMB07, the evolution efficiency  of the SIM module was higher.The strain was turned on to the mutagenic state by the addition of IPTG in the mid-log phase. After 18–24 h of IPTG induction, cells were spread onto LB plates containing an inhibitory concentration of volution . Althougn-butanol tolerance compared with its parent strain, SMB07/pML/pTL16  was only 14-fold. This result corresponds well with the observation that MMR genes in natural bacteria isolated from stressful environments are often deficient or show high sequence mosaicism [SIM has been considered with respect to microbial pathogenesis and antibiotic resistance, and tumor progression and chemotherapy resistance . Recentlosaicism –35. ThesumuD’ gene in the SOS response for error-prone DNA replication [ropE in heat-shock and envelope responses, and the transcription elongation factor nusA which recruits Pol IV (dinB) [+ reversion like umuD’ [recA was more efficient in elevating the SIM rate, with a 14-fold increase. The multifunctional RecA protein is responsible for DNA homologous recombination and activation of the SOS response and Pol V [+ reversion mutation [dinB) and the principle sigma transcription factor RopS (ropS) in the general stress response are also essential for SIM. Up-regulation of the error-prone DNA polymerase Pol IV (dinB) can tilt its competition with DNA polymerases Pol I, II, and III, thus producing stress-induced mutations [dinB or ropS decreased the Lac+ reversion mutation rate by 80–90 % [recA, dinB, and ropS was synergistic, as seen by the 32-fold increased SIM rate after expression of all three genes.Surprisingly, expression of the SIM accelerator genes which were up-regulated during SIM did not always give positive effects on the SIM rate. These include the lication , the sigV (dinB) . We suggke umuD’ . For thend Pol V . The impmutation . The trautations . Previou 80–90 % –42. In tEvolutionary engineering composed of mutagenesis followed by selection has been widely applied to improve microbial tolerance to environmental stresses. Although mutagenesis can be easily turned on and off by addition of foreign physical or chemical mutagens, the use of a SIM module in this study enables in vivo generation of genome-wide mutations in non-dividing cells and thus synchronizes the mutagenesis and selection process in a single-plate incubation step. Moreover, the SIM module avoids the use of any toxic mutagen to trigger the mutagenic state.mutL-defective strain to perform the SIM-based adaptive evolution, the most important advantage of using the SIM module is that it offers the feasibility of switching the mutagenic state back to the high-fidelity stage to stably maintain the improved phenotype at the end of evolution. However, the mutL gene is permanently deleted in SMB07 so that the strain is always in its mutagenic state. Cultivation of such a hypermutable strain will be problematic because mutations continue to emerge across the genome and the desired traits might disappear during long-term passages. Moreover, the SIM module expands the dynamic range of the SIM rates and provides the possibility of their fine-tuning. The strain SMB07 has a lower and fixed (82-fold increased) SIM rate, while the SIM module can reach a much higher (2923-fold increased) SIM rate. Different fluorescence values were generated by the toggle switch upon addition of different concentrations of IPTG or aTc , yielding pTL02 to pTL16  were added, and the incubation continued for 18–24 h. A 200-μL aliquot of culture was spread onto freshly prepared LB agar plates containing 25 μg/mL tetracycline, 100 μg/mL ampicillin, and an inhibitory concentration of n-butanol . These plates were incubated at 37 °C for 4–10 days in a sealed container. The colonies that gradually appeared were streaked on an LB plate with 25 μg/mL tetracycline, 100 μg/mL ampicillin, the same concentration of n-butanol, and 500 ng/mL aTc. After 2 days of incubation, the colonies exhibiting a rapid growth rate were selected for comparative analysis of growth capability in a sealed tube of LB medium containing 25 μg/mL tetracycline, 100 μg/mL ampicillin, the same concentration of n-butanol, and 500 ng/mL aTc. The best-performing strains were selected for the next cycle of adaptive evolution.Similar to a previously reported method , E. colin-butanol added. The OD600 value of cultures was detected using 96-well plates in a microtiter plate reader . The concentration of n-butanol that restricted the increase of OD600 by twofold or less was defined as the MIC.A fresh single colony was inoculated into 10 mL LB broth and cultured for 6–8 h at 37 °C, 200 rpm. Fresh LB medium in a sealed tube was then inoculated with a 1:50 aliquot of the culture and different concentrations of Additional file 1:Supplementary material.Table S1.E. coli strains and plasmids used in this study. Table S2. Sequences used in this study. Table S3. The Lac+ colonies of strains FC40 containing different plasmids on M9-lactose plates and their calculated SIM rates. Table S4. The Lac+ colonies of different strains on M9-lactose plates and their calculated SIM rates. Figure S1. Bistable control of the synthetic toggle switch using gfp as a reporter. Figure S2. Structures of the main plasmids and the induction strength of three ribosomal binding site sequences. Figure S3. The effect of different inducers on gfp expression in strain SMB07/pML/pTLCG. Figure S4.E. coli cells spread on LB agar plates with an inhibitory concentration of n-butanol can survive for 9 days and gradually formed colonies over 4–10 days.


Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river.blaCTX-M-15 mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology.River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate blaCTX-M-15 in UK river sediment; the prevalence of blaCTX-M-15 was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The blaCTX-M-15 gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river.We report the first examples of The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health. Few studies have demonstrated the impacts of liquid WWTP effluent on antibiotic resistance loads in rivers, particularly with reference to third-generation cephalosporin (3GC) resistance.3 The most common mechanism conferring resistance to 3GCs is the production of plasmid-mediated extended-spectrum β-lactamases (ESBLs), of which the most prevalent are the CTX-M enzymes encoded by blaCTX-M.4 Evidence suggests insertion sequence elements ISEcp1 and IS26 mobilized progenitors of blaCTX-M onto plasmids from the chromosome of Kluyvera species, a common rhizosphere organism.5 Subsequently, plasmid-borne blaCTX-M genes have disseminated throughout the Enterobacteriaceae and Gammaproteobacteria.6 Currently, there are >145 different genotypes of blaCTX-M (http://www.lahey.org/studies), which are often region specific, with blaCTX-M-15 being the most prevalent in humans worldwide and in the UK.4 Rivers are routinely used for the release of WWTP effluent and are a repository for sewage through storm drain overflow. A surveillance study found blaCTX-M-14 in UK rivers; however, to date, the most clinically important ESBL blaCTX-M-15 has not been found.7 Environmental reservoirs of antibiotic-resistant bacteria are likely to represent a significant exposure risk to humans, through direct contact or indirectly through contaminated drinking water or irrigation of crops. We hypothesize that waste water disposal methods are a contributing factor in resistance gene dissemination in rivers. The current study involves a comparative analysis of 3GC-resistant Enterobacteriaceae upstream and downstream of a WWTP effluent point.Growing evidence suggests anthropogenic activities such as agriculture contribute to environmental reservoirs of resistant bacteria that can directly or indirectly transfer to humans.Sampling took place in December 2009 and January 2011. Sediment core samples were taken from a river in the UK midlands at three sites in triplicate, 300, 600 and 900 m upstream of a WWTP and three sites in triplicate, 300, 600 and 900 m downstream of a WWTP. The treatment plant served ∼500 000 people and processed >120 million litres of raw sewage per day using a primary settlement tank, secondary activated sludge treatment and final tertiary filtration. Upstream of the treatment plant, geospatial analyses had indicated no other WWTP for ≥10 km. All samples were immediately stored at 4°C and processed within 24 h.Escherichia coli and pink colonies indicated other coliforms termed presumptive coliforms excluding E. coli (PCEs). Reference strains of E. coli, Klebsiella oxytoca, Citrobacter freundii, Pseudomonas fluorescens and Aeromonas media were used to evaluate the performance of Chromocult at 30°C.Sediment from one of each downstream sample site  was pooled in equal parts  and resuspended in 1 mL of PBS buffer. This was repeated for upstream sediment samples. In total, each of the triplicate sediment samples was pooled to form three downstream samples and three upstream samples. Chromocult Coliform Agar (Merck) was prepared in accordance with the manufacturer's instructions and amended with cefotaxime (2 mg/L) or ceftazidime (16 mg/L). Downstream and upstream samples were plated (200 μL) in triplicate for each antibiotic and unamended Chromocult, before incubating for 24 h at 30°C. Viable plate counts were taken; blue colonies indicated presumptive E. coli were picked and streaked to obtain pure cultures. The number of isolates obtained for each site differed due to different resistance gene prevalences between sample sites.PCEs and 8MICs of cefotaxime (1–2048 mg/L) and imipenem (1–32 mg/L) were determined using a broth microdilution method based on CLSI and EUCAST standards as previously described.Isolates were incubated overnight at 30°C in Luria broth (LB) and DNA was extracted using a Nucleospin Blood Kit (Macherey-Nagel) in accordance with the manufacturer's instructions.9 Further identification of Enterobacteriaceae was performed by partial sequencing of dnaJ as previously described.10Aeromonas spp. were identified using partial sequencing of gyrB.11E. coli strains were typed using the Achtman scheme.12Bacteria were first identified by sequencing PCR products obtained using the universal 27F and 1525R 16S rRNA primers.bla genes, including blaTEM, blaSHV and blaCTX-M, was performed as previously described.13PCR amplification of blaCTX-M was performed with PCR as previously described.14Characterization of the regions flanking JAC OnlineSupplementary data at )15 and newly designed primers  were used to detect duplications (please see the JAC OnlineSupplementary data at ).Further analysis of flanking regions unidentified by conventional PCR was done by modifying the two-step gene-walking method (please see the E. coli DH10B (StrR) with induced rifampicin resistance was used as a recipient strain for solid conjugal mating assays with positive blaCTX-M-15 strains as donors. Transconjugants were selected using LB plates amended with streptomycin (100 mg/L), rifampicin (100 mg/L) and cefotaxime (2 mg/L). Positive transconjugants were confirmed using the PCR primer pair CTX-F and CTX-R.blaCTX-M-15-positive strains were identified using a PCR-based method.16 Strains with identical replicon types were further analysed using restriction fragment length polymorphism (RFLP) (please see the JAC OnlineSupplementary data at ).Plasmid replicon types in t-test. Proportions were compared using Fisher's exact test.All statistics were performed using Genstat 15th edition SP1 . For comparison of means, log counts were checked for normal distribution using the Shapiro–Wilk test followed by analysis using a paired-sample E. coli and PCEs in the river sediment downstream of effluent discharge in both 2009 and 2011. No significant difference was recorded in numbers of 3GC-resistant E. coli in downstream samples between 2009 and 2011; however, there was a significant increase in numbers of 3GC-resistant PCEs in upstream samples between 2009 and 2011 . The mean average number of total coliforms in 2011 was 4 × 105/g of wet sediment downstream and 2 × 105/g of wet sediment upstream. The mean average number of E. coli in 2011 was 8 × 103/g of wet sediment downstream and 4 × 103/g of wet sediment upstream. From this, we can calculate that 0.95% of E. coli were resistant to 3GCs downstream compared with 0.13% of E. coli upstream. Similarly, for coliforms there were 0.079% resistant downstream compared with 0.042% upstream.There was a significant increase in the numbers of 3GC-resistant presumptive E. coli (n = 41) were confirmed as E. coli by sequencing dnaJ PCR products (Table n = 19) were also identified using dnaJ  analysis of E. coli isolates revealed uncharacterized sequence types (STs) particularly from upstream samples (80%), indicating the existence in this environment of novel STs. Downstream of the WWTP, the human-associated ST3103 and ST38 were codominant in 2009, but neither of these STs was detected in 2011 samples, which were dominated by the well-recognized human disease-associated types ST131 (20%) and ST167 (25%) [Table JAC Online)Table S1  both came from upstream of the WWTP. The CTXD-F and CTXD-R primers allowed for detection of blaCTX-M-15 repeats found in Groups K, L and R; however, several groups were unresolved even after two-step gene walking, due to multiple copies of blaCTX-M-15 and repeat regions. Aside from Group A, Group I was the only other group recorded in both 2009 and 2011, but in E. coli in 2009 and K. oxytoca in 2011.A total of 11 different genetic contexts were found in association with blaCTX-M-15 flanking regions, such as IS1, putative phage proteins, toxin genes and resistance genes to other antibiotics ; however, promoter analysis  revealed that all but one of the contexts shared the same promoter region of blaCTX-M-15 as Group A. One E. coli strain had a high level of resistance to imipenem (>32 mg/L), though this was not conferred through blaCTX-M-15 (Table S1).Isolates containing seven of the new genetic contexts had MICs of cefotaxime >1024 mg/L. Significantly higher MICs were characteristic of isolates found in downstream samples compared with upstream samples (Table S1). IncF was the most prevalent replicon type both downstream and upstream of the WWTP. The same genetic context was regularly recorded in strains carrying different replicon types, with the Group A context being associated with the most diverse range of replicons . Group N was carried by the same plasmid upstream and downstream of the WWTP across different families; initially upstream in A. media and downstream in K. oxytoca and E. coli.In both years, a number of different replicon types were associated with CTX-M-carrying strains .Conjugation experiments revealed all but two of the 17 We demonstrated that even with this level of treatment, WWTP effluent has a significant impact on numbers of 3GC-resistant bacteria in river sediment communities. As well as an increase in the total numbers of 3GC-resistant bacteria, the WWTP had an impact on the prevalence of 3GC resistance in bacteria with a 7-fold increase in the prevalence of 3GC-resistant E. coli. The cause of 3GC resistance predominantly resulted from the dissemination of blaCTX-M-15. This is the first report of blaCTX-M-15 in UK river waters and represents a worrying trend as this gene is the most common ESBL in E. coli and Klebsiella spp. causing clinical disease.4 Novel hosts were isolated across the Gammaproteobacteria, including Citrobacter braakii, A. media and P. fluorescens, none of which has previously been reported as a carrier of blaCTX-M-15. Many of the resistant bacteria were pathogens, such as E. coli ST131, ST167 and ST38, C. freundii and K. oxytoca. In particular, the finding of blaCTX-M-15 in the pandemic pathogen E. coli ST131 as a viable and significant reservoir in environmental samples represents a serious threat to human health. This supports recent findings of the threat that rivers pose to human health highlighted by a study in which one-third of people swimming in areas of the River Thames suffered gastrointestinal illness.18 In addition, we report the first finding of an imipenem-resistant E. coli in a UK river, an indication of the emerging spread of carbapenem resistance in the environment, which is a great cause for concern.Tertiary treatment by WWTPs is the most rigorous level of waste water treatment in the UK as set out by the Water Services Regulation Authority (Ofwat).blaCTX-M-15 carriage and hypothesize that such an unprecedented diversity can be attributed to the direct introduction of bacteria by WWTP effluent possibly combined with in situ selection either in the river or WWTP. Selection is likely to be aided by antibiotic and detergent residues that have previously been detected in WWTP effluent as well as the high density of bacteria present in WWTPs, which will facilitate cell-to-cell contact.19 This hypothesis is supported by plasmid analyses as replicon typing revealed eight types present in isolates carrying blaCTX-M-15. Of particular concern is the frequency (46%) at which multiple plasmid replicons were colocalized in one isolate. This would allow for interplasmid transfer of blaCTX-M-15 through transposition and homologous recombination and each different genetic context of blaCTX-M-15 may be indicative of a transfer event.20We demonstrated that there was high genetic diversity in 21 The extensive mobility of plasmids was further emphasized by the recovery of identical plasmids in diverse backgrounds. Of concern was the pool of plasmids shared between hosts regarded as clinical bacteria and those regarded as indigenous to the river environment.The carriage of multiple plasmid incompatibility groups will contribute to higher conjugation rates. This resulted in conjugation frequencies that were higher than reported for similar studies conducted with clinical strains and plasmids.blaCTX-M-15 flanking regions, including E. coli ST131-carrying Group Q. The prevalence of human-associated bacteria with a high diversity of blaCTX-M-15 flanking regions downstream of the WWTP supports the hypothesis that community carriage is more extensive than currently thought.22 The change in prevalence of E. coli STs between 2009 and 2011, with ST131 becoming one of the most dominant STs, is likely a reflection of the clonal spread of this ST in the human population.22We have demonstrated repeated evidence of the significant introduction of clinically relevant ESBL-producing bacteria by WWTP effluent into a UK river. Many of the pathogens had novel blaCTX-M-1 in UK cattle, chickens, turkeys and most recently dogs.23 The gene blaCTX-M-15 has been detected throughout Europe in companion animals and a diverse range of wild birds.24 Whilst it is not possible to determine the direction of spread from humans to animals, the significant environmental reservoir in rivers will impact both. In conclusion, we report a reservoir of blaCTX-M-15 in a UK river with clear evidence of extensive recombination of the gene within plasmid populations. A growing environmental reservoir presents a risk to human health, with evidence implicating WWTP effluent as a major contributor to the formation of this reservoir.An increase in the number of 3GC-resistant coliforms upstream between 2009 and 2011 is potentially a consequence of faecal contamination from surrounding farm environments. This timescale coincides with the detection of the ESBL gene Further research is needed into sewage treatment systems that result in minimal introduction of resistant bacteria and selecting agents such as antibiotic residues and quaternary ammonium compounds. Stricter regulations and higher levels of treatment are needed if we are to halt the rise of antibiotic resistance in the environment.Natural Environment Research Council (grant NE/E004482/1). G. C. A. A. was supported by a BBSRC studentship. W. H. G. has been supported by the ERDF and ESF since moving to the University of Exeter.This work was supported by the None to declare.G. C. A. A., W. H. G. and E. M. W. designed the research, G. C. A. A. performed the research, all authors analysed the data and all authors contributed to writing the paper.JAC Online .